(a) Field of the Invention
The present invention relates to a method of separating and purifying 13-dehydroxybaccatin III and 10-deacetylpaclitaxel with high purity of 90% or higher purity, preferably 99.5% or higher purity, from taxane-containing materials, and the thusly obtained highly pure 13-dehydroxybaccatin III and 10-deacetylpaclitaxel can be used as semi-synthetic precursors of paclitaxel and/or docetaxel that are useful anticancer agents.
(b) Description of the Related Art
13-dehydroxybaccatin III and 10-deacetylpaclitaxel are precursors that can be converted into paclitaxel or docetaxel by chemical reactions. Since paclitaxel was a result from large-scale screening programs for antitumoral substances by U.S. National Cancer Institute in 1960s, it has been known as one of the most important anticancer substance for ovarian cancer, breast cancer, etc., and therefore, it has been widely used with the approval by U.S. Food and Drug Administration (FDA) in 1992. However, as this substance is isolated and purified from the bark of yew, which contains substance in a very small amount as low as 0.02%, the separation and purification of the substance are very costly, and furthermore, it becomes one of the factors in the destruction of resource and ecosystem.
In order to overcome such drawbacks, methods of semi-synthesizing paclitaxel by using baccatin III or 10-deacetylbaccatin III obtained from the leaves of yew and methods for mass production thereof based on cell culture technique of yew have been developed.
In addition, 13-dehydroxybaccatin III and 10-deacetylpaclitaxel can be used as important precursors for paclitaxel [Rao K V, Bhakuni R S, Johnson J, Oruganti R S., Synthesis and evaluation of some 10-mono- and 2′,10-diesters of 10-deacetylpaclitaxel. J. med. Chem., 1995, 38, 3411-3414.; Ojima I, Sun C M, Zucco M, Park Y H, Duclos O, Kuduk S, A highly efficient route to taxoter by the β-lactam synthon method. Tetrahedron Lett., 1993, 34, 4149-4152.; Commercon A, Bezard D, Bernard F, Bourzat J D., Improved protection and esterification of a precursor of the Taxotere and taxol side chains. Tetrahedron Lett., 1992, 33, 5185.; Holton R, “Method for preparation of Taxol using an oxazinone.” European Patent Application 400, 971(1990), U.S. Pat. No. 5,015,744(1991); “Method for preparation of taxol using β-lactam.” European Patent Application 428,375(1991), U.S. Pat. No. 5,175,315(1992)], and these substances are co-produced along with paclitaxel during the course of production of paclitaxel by yew or yew cell cultures.
Separation and purification methods for 13-dehydroxybaccatin III and 10-deacetylpaclitaxel have not been widely known in documents, and in particular, no records can be found in the case of 13-dehydroxybaccatin III. In the case of 10-deacetylpaclitaxel, U.S. Pat. No. 5,475,120 discloses methods for extraction and purification of paclitaxel and several derivatives from yew. The process disclosed in this patent comprises obtaining several fractions using extraction, partition, and reverse phase chromatography using C-18 (15-35 micron) of preparative scale and then purifying useful components including paclitaxel, 10-deacetylbaccatin III and 10-deacetylpaclitaxel from each fraction. However, the C-18 resins are expensive as they are 15-35 micron, and the applied specimens are extracts, which can readily contaminate the C-18 resins, and thus, their durability can be shortened. Besides, as there have been no evaluation information relating to purity and yields according to each purification step, it is difficult to understand the efficiency of the process.
Further, Erik L. M. et al. published research results about the content analysis of paclitaxel, 10-deacetylpaclitaxel, baccatin III, etc. by using HPLC after extraction using organic solvents from several Taxus species and pre-treatment using solid phase extraction column (Phytochemistry, 53(2000) 383-389), but this method is suitable only for quantity analysis using HPLC, not for isolating useful components. No detailed information on mass purification for 10-deacetylpaclitaxel have yet been reported.
U.S. Pat. No. 6,002,025 discloses a method for purification by fraction collecting taxanes such as 10-deacetylpaclitaxel, baccatin III and paclitaxel of 50% or higher purity using phenylalkyl column and preparative HPLC; however, it is not a process which is related to extraction and isolation from yew or cultured yew cells. That is, the disclosed process is restricted to purifying partially-purified taxanes by using preparative HPLC.
Therefore, the development of efficient isolation and purification methods of 13-dehydroxybaccatin III and 10-deacetylpaclitaxel of high purity from taxane-containing materials is in need.